EP1123388B1 - Transgenic c. elegans as a model organism for research into alzheimer's disease - Google Patents

Transgenic c. elegans as a model organism for research into alzheimer's disease Download PDF

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EP1123388B1
EP1123388B1 EP99947485A EP99947485A EP1123388B1 EP 1123388 B1 EP1123388 B1 EP 1123388B1 EP 99947485 A EP99947485 A EP 99947485A EP 99947485 A EP99947485 A EP 99947485A EP 1123388 B1 EP1123388 B1 EP 1123388B1
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elegans
nucleotide sequence
app
peptide
seq
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EP1123388A1 (en
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Giesela Peraus
Edmund Hoppe
Ralf Baumeister
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Sanofi Aventis Deutschland GmbH
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/033—Rearing or breeding invertebrates; New breeds of invertebrates
    • A01K67/0333—Genetically modified invertebrates, e.g. transgenic, polyploid
    • A01K67/0335—Genetically modified worms
    • A01K67/0336—Genetically modified Nematodes, e.g. Caenorhabditis elegans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4711—Alzheimer's disease; Amyloid plaque core protein
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

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  • the present invention relates to the use of a transgenic C. elegans containing a transgene which codes for the amyloid precursor protein (APP) or a part thereof for the identification and / or characterization of substances which inhibit ⁇ -secretase activity or which express the ⁇ -Secretase activity, or to validate an ⁇ - and / or a ⁇ secretion activity or to identify a ⁇ -secretase activity in C. elegans.
  • APP amyloid precursor protein
  • Alzheimer's disease is a neurodegenerative disease of the brain that is accompanied at the cellular level by a massive loss of neurons in the limbic and cerebral cortex.
  • protein deposits so-called plaques
  • a ⁇ peptide The most abundant protein in these plaques is a 40 to 42 amino acid peptide termed the A ⁇ peptide.
  • This peptide is a cleavage product of a much larger protein of 695 to 751 amino acids, the so-called amyloid precursor protein ( A myloid P recursor P rotein, APP).
  • APP is an integral transmembrane protein that traverses the lipid bilayer once. By far the largest part of the protein is extracellular, while the shorter C-terminal domain is directed into the cytosol ( FIG. 1 ).
  • the A ⁇ peptide is in FIG. 1 shown in dark gray. About two-thirds of the A ⁇ peptide is from the extracellular domain and about one-third from the transmembrane domain of APP.
  • APPsec secreted APP
  • Cleavage occurs at a site of the amino acid sequence of APP that is within the amino acid sequence of the A ⁇ peptide (after amino acid residue 16 of the A ⁇ peptide). Proteolysis of APP by the ⁇ -secretase thus precludes the formation of the A ⁇ peptide.
  • the A ⁇ peptide can only be formed from APP by an alternative processing route. It is postulated that two more proteases are involved in this processing pathway, with one protease termed ⁇ -secretase cleaving at the N-terminus of the A ⁇ peptide in the APP and the second protease, termed ⁇ -secretase , the C-terminus of the Aß-peptide releases ( Kang, J. et al., Nature, 325, 733 ) ( FIG. 1 ).
  • a ⁇ peptide is a critical factor in the onset of Alzheimer's disease.
  • a neurotoxicity of Aß fibrils in cell culture is postulated ( Yankner, BA et al., (1990) Proc Natl Acad Sci USA, 87, 9020 ).
  • the neuropathology characteristic of Alzheimer's disease already occurs at the age of 30 years. It is assumed that overexpression of APP is followed by increased conversion into the A ⁇ peptide ( Rumble, B. et al., (1989), N. Engl. J. Med., 320, 1446 ).
  • Aß peptide Perhaps the strongest indication of the central role of the Aß peptide are the familial forms of Alzheimer's disease. Here are mutations in the APP gene around the area of the ß- and ⁇ -secretase interfaces or in two other AD-associated genes (presenilines), which lead to a significant increase in Aß production in cell culture ( Scheuner, D. et al., (1996) Nature Medicine, 2, 864 ).
  • C. elegans has already been used as a model organism in Alzheimer's disease, however, this work does not relate to the processing of APP into the A ⁇ peptide.
  • the presenilins are transmembrane proteins that traverse the membrane 6-8 times. They are of great importance in familial Alzheimer cases, as certain mutations in the Presenilingen lead to Alzheimer's disease. It has been shown that C.
  • elegans homologues to the human presenilines exist, wherein the function of presenilins is conserved in humans and worms ( Levitan D, Greenwald I (1995) Nature 377, 351 ; Levitan et al (1996) Proc Natl Acad Sci USA, 93, 14940 ; Baumeister R (1997) Genes & Function 1, 149 ; Xiajun Li and Iva Greenwald (1997) Proc Natl Acad Sci USA, 94, 12204 ).
  • C. elegans C. elegans
  • Apl-1 does not have a region homologous to the amino acid sequence of the A ⁇ peptide, therefore C. elegans does not have an endogenous A ⁇ peptide ( Daigle I, Li C (1993) Proc Natl Acad Sci USA, 90 (24), 12045 ).
  • a ⁇ peptide (not that of an A ⁇ precursor protein) in C. elegans became by CD Link, Proc Natl Acad Sci USA (1995) 92, 9368 described.
  • transgenic worms were produced expressing an A ⁇ 1-42 peptide (ie the A ⁇ peptide consisting of 42 amino acids) under the control of the muscle specific promoter unc54 as a fusion protein with a synthetic signal peptide.
  • muscle-specific protein deposits that reacted with anti- ⁇ -amyloid antibodies were detected.
  • Further work (eg, C. Link et al., Personal communication) relates to studies of the aggregation and toxicity of the A ⁇ peptide in the C. elegans model system.
  • the subject of the present invention therefore relates to the following use.
  • the nucleotide sequence encodes the 100 carboxy-terminal amino acids of APP starting with the sequence of the A ⁇ peptide and ending with the carboxy-terminal amino acid of APP encoded (C100 fragment).
  • the APP is preferably one of the isoforms APP695 (695 amino acids), APP751 (751 amino acids), APP770 (770 amino acids) and L-APP. All isoforms are formed by alternative splicing from the same APP gene. In APP695, exons 7 and 8 were removed by splicing, whereas in APP751 only exon 8 is absent and present in APP770 exons 7 and 8. In addition, there are other splice forms of APP, where the exon 15 is removed by splicing. These forms are referred to as L-APP and are also present in the forms spliced to exons 7 and 8.
  • the transgene contains the nucleotide sequence SEQ ID NO .: 1 or a part thereof.
  • the transgene contains a further coding nucleotide sequence located at the 5 'end of the nucleotide sequence encoding APP or a portion thereof.
  • the further nucleotide sequence codes for a signal peptide or a part thereof, for example for the APP signal peptide (SP) with the amino acid sequence SEQ ID NO .: 9 or a part thereof.
  • the sequence from the N-terminus of the A ⁇ peptide to the C-terminus of APP consists of 99 amino acids.
  • the APP signal peptide consists of 17 amino acids.
  • one or more "spacer amino acids” are preferably inserted between these two parts of the fusion product, preferably an amino acid is inserted , for example leucine.
  • the transgene contains the nucleotide sequence SEQ ID NO .: 2 or a part thereof and / or the nucleotide sequence SEQ ID NO .: 3 or a part thereof.
  • the transgene may contain one or more other non-coding and / or one or more additional coding nucleotide sequences.
  • the transgene may contain, as a further non-coding nucleotide sequence, a sequence of an intron of the APP gene, for example a sequence which originates from the intron 42 bp of the APP gene and has the sequence SEQ ID NO .: 4.
  • the object of the invention is a transgene which contains the nucleotide sequence SEQ ID NO .: 5.
  • the transgene also contains one or more gene regulatory sequences for the regulated expression of the encoded protein, preferably a constitutive or a regulatable promoter.
  • the promoter may be active in C. elegans neuronal, muscular or dermal tissue or ubiquitous in C. elegans.
  • a promoter may be selected from the range of C. elegans promoters unc-54, hsp16-2, unc-119, goa-1 and sel-12.
  • the transgene contains a promoter having the nucleotide sequence SEQ ID NO .: 6.
  • the transgene contains the nucleotide sequence SEQ ID NO .: 7.
  • the transgene may be present in a vector, for example in an expression vector.
  • a recombinant expression vector may contain the nucleotide sequence of SEQ ID NO: 8.
  • the invention also describes the production of an expression vector, wherein a transgene is integrated into a vector by known methods.
  • the invention describes the use of an expression vector for the production of a transgenic cell, which cell is part of a non-human organism, e.g. C. elegans, can be.
  • the invention also describes the preparation of the transgene, wherein suitable partial sequences are ligated in the appropriate order and in the correct reading frame, if appropriate with the insertion of linkers.
  • the invention describes the use of the transgene, eg for the production of a transgenic cell, which cell may be part of a non-human organism.
  • the cell may be a C. elegans cell.
  • a particular embodiment of the invention describes a transgenic C. elegans containing the transgene.
  • the transgene may also be present in the C. elegans in an expression vector.
  • the transgene may be present in the C. elegans intra- and / or extrachromosomal. Intra- and / or extrachromosomal, one or more transgenes or expression vectors containing the transgene, as
  • a transgenic cell or transgenic organism additionally contains another expression vector containing a nucleotide sequence encoding a marker, which marker is either a temperature-sensitive or a phenotypic marker.
  • the marker may be a visual or a behavioral phenotypic marker.
  • fluorescent labels e.g. GFP (Green Fluorescent Protein) or EGFP (Enhanced Green Fluorescent Protein)
  • marker genes encoding a dominant mutated form of a particular protein, e.g. for a dominant RoI6 mutation or marker sequences encoding antisense RNA, e.g. for the antisense RNA of Unc-22.
  • one or more copies of the transgene and / or the expression vector are present in the germ cells and / or the somatic cells of the transgenic C. elegans and, if appropriate, a further expression vector.
  • the invention also describes methods for producing a transgenic C. elegans, wherein a transgene and / or an expression vector, if appropriate in the presence of a further expression vector which contains a nucleotide sequence which codes for a marker, is microinjected into the germinal cells of a C. elegans.
  • the invention also describes the use of a transgenic C. elegans, for example for expression of an SP-C100 fusion protein.
  • An object of the invention uses a Sp-C100 fusion protein having the amino acid sequence SEQ ID NO .: 10.
  • the invention relates to the use of a transgenic C. elegans for the identification of ⁇ -secretase activity and / or ⁇ -secretase activity in C. elegans, for use in methods for the identification and / or characterization of substances having the ⁇ -secretase activity use in methods for the identification and / or characterization of substances that increase ⁇ -secretase activity, use in methods for the identification and / or characterization of substances used as active ingredients for the treatment and / or prevention of Alzheimer's disease can be.
  • C. elegans the nematode Caenorhabditis elegans
  • This worm is excellently suited for genetic studies and has therefore been widely used in the past to detect universally important processes, e.g. to study programmed cell death, neuronal guidance, and RAS / MAP kinase signaling (Riddle, D.L. et al., 1997).
  • FIG. 1 shows the amyloid precursor protein (isoform APP695 and isoforms APP770 and APP751, respectively) and secretase cleavage products.
  • C100 consists of the A ⁇ sequence and the C-terminus of APP ( Shoji, M et al., (1992) Science 258, 126 ).
  • Unc-119-SP-C100 has 5112 base pairs.
  • Example 1 Preparation of an expression vector containing the transgene.
  • the base vector pBY103 consists of the vector backbone pPD49.26 described in " Caenorhabditis elegans: Modern Biological Analysis of an Organism "(1995) Ed. Epstein et al., Vol 48, pp. 473 into which the unc-119 promoter ( Maduro et al. Genetics (1995), 141, p.
  • the plasmid unc-119-SP-C100 was prepared by KpnI / SacI cut and subsequent cloning of the LC99 fragment from pSKLC1-99 (Shoji et al. (1992) into the pBY103.
  • the unc-119 SP-C100 construct was microinjected into the gonads of young adult hermaphrodites using a microinjection system.
  • the DNA concentration was about 20 ng / ⁇ l.
  • a marker plasmid was injected. It is the ttx3-GFP plasmid that encodes the Green Fluorescent Protein under the control of the ttx3 promoter.
  • the activity of the ttx3 promoter is specific for certain head neurons of C. elegans, the so-called AIY neurons, which play a role in the thermotaxis of the worm.
  • Successfully injected worms exhibit green fluorescence in the head of the AIY neurons when excited with light at a wavelength of about 480 nm. Such nematodes could be detected.
  • C100 transgenic worms show a green fluorescence in the AIY neurons of the head area after excitation with light of the wavelength of 480 nm. Since green fluorescence was again detectable in the head neurons in the offspring of the worms, it can be assumed that the plasmids are pathway-friendly. However, the penetrance is not 100%, suggesting that the long tandam arrays of ttx3-GFP marker DNA and unc-119-SP-C100 are more extrachromosomally integrated than in the genome.
  • 96-well plates are first incubated with the monoclonal antibody clone 6E10 (SENETEK PLC., MO, USA), which reacts specifically with the A ⁇ peptide (amino acid 1-17), and then with worm extracts from transgenic worms or control worms.
  • the detection of the A ⁇ peptide takes place with the aid of the monoclonal A ⁇ antibody 4G8 (SENETEK PLC., MO, USA), which recognizes amino acids 17-24 in the A ⁇ peptide and is labeled with biotin.
  • the detection is carried out via the alkaline phosphatase reaction with a corresponding antibody which is directed against biotin.
  • the digestion of the worms implies detergent treatment, nitrogen shock freezing, sonication, and rupture of the cells with glass beads.
  • the ELISA signal from the experiment described above may underlie both weak expression of the A ⁇ peptide and that of the C100 precursor protein, since the corresponding epitopes are present in both proteins.
  • the expression of the A ⁇ peptide could be detected specifically:
  • Aß-specific antibodies that do not react with the C 100 precursor would have to be used in an A ⁇ sandwich ELISA.
  • an A ⁇ -specific antibody could be a monoclonal antibody that specifically recognizes the C-terminal end of the A ⁇ form, which is composed of 40 or 42 amino acids.
  • the A ⁇ peptide could be detected in the Western blot using monoclonal antibodies 4G8 and 6E10 and then discriminated from the larger C100 precursor due to its molecular weight of 4kD.

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Abstract

The present invention relates to a transgenic C. elegans which expresses an amyloid precursor protein (APP) or a part thereof, to the transgene itself, to the protein encoded by the transgene, and also to a process for preparing the transgenic C. elegans and to its use.

Description

Die vorliegende Erfindung betrifft die Verwendung eines transgenen C. elegans enthaltend ein Transgen, welches für da Amyloid Vorläufer Protein (APP) oder einen Teil davon kodiert, zur Identifizierung und/oder Charakterisierung von Substanzen, die die γ-Sekretase Aktivität hemmen oder die die α-Sekretase Aktivität erhöhen, bzw. zur Validierung einer α- und/oder einer γ-Sekretionsaktivität bzw. zur Identifizierung einer γ-Sekretase Aktivität in C. elegans.The present invention relates to the use of a transgenic C. elegans containing a transgene which codes for the amyloid precursor protein (APP) or a part thereof for the identification and / or characterization of substances which inhibit γ-secretase activity or which express the α -Secretase activity, or to validate an α- and / or a γ secretion activity or to identify a γ-secretase activity in C. elegans.

Bei der Alzheimer'schen Krankheit (Morbus Alzheimer) handelt es sich um eine neurodegenerative Erkrankung des Gehirns, die auf zellulärer Ebene mit einem massiven Verlust von Neuronen im limbischen System und im cerebralen Cortex einher geht. Auf molekularer Ebene lassen sich in den betroffenen Gehirnarealen Proteinablagerungen, sogenannte Plaques, nachweisen, die ein wesentliches Charakteristikum der Alzheimer'schen Krankheit darstellen. Das am häufigsten in diesen Plaques vorkommende Protein ist ein 40 bis 42 Aminosäuren großes Peptid, das als Aß-Peptid bezeichnet wird. Bei diesem Peptid handelt es sich um ein Spaltprodukt eines wesentlich größeren Proteins von 695 bis 751 Aminosäuren, dem sogenannten Amyloidvorläuferprotein (Amyloid Precursor Protein; APP).Alzheimer's disease (Alzheimer's disease) is a neurodegenerative disease of the brain that is accompanied at the cellular level by a massive loss of neurons in the limbic and cerebral cortex. On the molecular level, protein deposits, so-called plaques, can be detected in the affected brain areas, which are an essential characteristic of Alzheimer's disease. The most abundant protein in these plaques is a 40 to 42 amino acid peptide termed the Aβ peptide. This peptide is a cleavage product of a much larger protein of 695 to 751 amino acids, the so-called amyloid precursor protein ( A myloid P recursor P rotein, APP).

APP ist ein integrales Transmembranprotein, das die Lipiddoppelschicht einmal durchquert. Der weitaus größte Teil des Proteins liegt extrazellulär, während die kürzere C-terminale Domäne in das Cytosol gerichtet ist (Figur 1). Das Aß-Peptid ist in Figur 1 dunkelgrau dargestellt. Etwa zwei Drittel des Aß-Peptids stammen aus der extrazellulären Domäne und etwa ein Drittel aus der Transmembrandomäne von APP.APP is an integral transmembrane protein that traverses the lipid bilayer once. By far the largest part of the protein is extracellular, while the shorter C-terminal domain is directed into the cytosol ( FIG. 1 ). The Aβ peptide is in FIG. 1 shown in dark gray. About two-thirds of the Aβ peptide is from the extracellular domain and about one-third from the transmembrane domain of APP.

Neben dem membranständigen APP läßt sich eine sekretierte Form des Amyloid Vorläuferproteins nachweisen, die aus der großen Ektodomäne des APPs besteht und als APPsec ("sekretiertes APP") bezeichnet wird. APPsec entsteht aus APP durch proteolytische Spaltung, die durch die ∀-Sekretase erfolgt. Die proteolytischeIn addition to the membrane-bound APP, a secreted form of the amyloid precursor protein can be detected, which consists of the large ectodomain of the APP and is referred to as APPsec ("secreted APP"). APPsec arises from APP by proteolytic cleavage, which occurs through the ∀-secretase. The proteolytic

Spaltung findet an einer Stelle der Aminosäuresequenz von APP statt, die innerhalb der Aminosäuresequenz des Aß-Peptids liegt (nach Aminosäurerest 16 des Aß-Peptids). Eine Proteolyse von APP durch die α-Sekretase schließt damit die Bildung des Aß-Peptids aus.Cleavage occurs at a site of the amino acid sequence of APP that is within the amino acid sequence of the Aβ peptide (after amino acid residue 16 of the Aβ peptide). Proteolysis of APP by the α-secretase thus precludes the formation of the Aβ peptide.

Das Aß-Peptid kann also nur auf einem alternativen Prozessierungsweg aus APP gebildet werden. Es wird postuliert, daß an diesem Prozessierungsweg zwei weitere Proteasen beteiligt sind, wobei die eine Protease, die als ß-Sekretase bezeichnet wird, am N-Terminus des Aß-Peptids im APP schneidet und die zweite Protease, die als γ-Sekretase bezeichnet wird, den C-Terminus des Aß-Peptids freisetzt ( Kang, J. et al., Nature, 325, 733 ) (Figur 1).Thus, the Aβ peptide can only be formed from APP by an alternative processing route. It is postulated that two more proteases are involved in this processing pathway, with one protease termed β-secretase cleaving at the N-terminus of the Aβ peptide in the APP and the second protease, termed γ-secretase , the C-terminus of the Aß-peptide releases ( Kang, J. et al., Nature, 325, 733 ) ( FIG. 1 ).

Bisher konnte keine der drei Sekretasen bzw. Proteasen (α-Sekretase, β-Sekretase, γ-Sekretase) identifiziert werden. Die Kenntnis der Sekretasen ist jedoch von großem Interesse, insbesondere im Rahmen von Untersuchungen zur Alzheimer'schen Krankheit und zur Identifizierung der beteiligten Proteine, die dann wiederum als Targets in weiterführenden Studien eingesetzt werden können, da zum einen die Inhibition der ß - und insbesondere der γ-Sekretase zu einer Reduktion der Aß-Produktion führen könnte, zum anderen eine Aktivierung der α-Sekretase die Prozessierung von APP in APPsec steigern und damit gleichzeitig die Entstehung des Aß-Peptids reduzieren würde.So far, none of the three secretases or proteases (α-secretase, β-secretase, γ-secretase) could be identified. However, the knowledge of the secretases is of great interest, in particular in the context of studies on Alzheimer's disease and the identification of the proteins involved, which in turn can be used as targets in further studies, as on the one hand, the inhibition of ß - and especially the On the other hand, activation of the α-secretase would increase the processing of APP into APPsec and thereby simultaneously reduce the formation of the Aβ peptide.

Es gibt viele Hinweise darauf, daß das Aß-Peptid bei dem Auftreten der Alzheimer'schen Krankheit ein entscheidender Faktor ist. Unter anderem wird eine Neurotoxizität von Aß-Fibrillen in Zellkultur postuliert ( Yankner, B.A. et al., (1990) Proc Natl Acad Sci USA,87, 9020 ). Auch tritt bei Patienten mit Down Syndrom, bei denen APP in einer zusätzlichen Kopie vorkommt die für den Morbus Alzheimer charakteristische Neuropathologie bereits im Alter von 30 Jahren auf. Dabei wird angenommen, daß der Überexpression von APP eine erhöhte Umsetzung in das Aß-Peptid folgt ( Rumble, B. et al., (1989), N. Engl. J. Med., 320, 1446 ).There is much evidence that the Aβ peptide is a critical factor in the onset of Alzheimer's disease. Among other things, a neurotoxicity of Aß fibrils in cell culture is postulated ( Yankner, BA et al., (1990) Proc Natl Acad Sci USA, 87, 9020 ). Also, in patients with Down syndrome, where APP is present in an additional copy, the neuropathology characteristic of Alzheimer's disease already occurs at the age of 30 years. It is assumed that overexpression of APP is followed by increased conversion into the Aβ peptide ( Rumble, B. et al., (1989), N. Engl. J. Med., 320, 1446 ).

Den vielleicht stärksten Hinweis auf die zentrale Rolle des Aß-Peptids stellen die familiären Formen der Alzheimer'schen Krankheit dar. Hier finden sich Mutationen im APP-Gen um den Bereich der ß- und γ-Sekretaseschnittstellen oder in zwei weiteren AD-assoziierten Genen (Presenilinen), die in Zellkultur zu einer wesentlichen Erhöhung der Aß-Produktion führen ( Scheuner, D. et al., (1996), Nature Medicine, 2, 864 ).Perhaps the strongest indication of the central role of the Aß peptide are the familial forms of Alzheimer's disease. Here are mutations in the APP gene around the area of the ß- and γ-secretase interfaces or in two other AD-associated genes (presenilines), which lead to a significant increase in Aß production in cell culture ( Scheuner, D. et al., (1996) Nature Medicine, 2, 864 ).

C. elegans wurde bereits als Modellorganismus bei der Alzheimer'schen Krankheit eingesetzt, jedoch beziehen sich diese Arbeiten nicht auf die Prozessierung von APP in das Aß-Peptid. Einige Arbeiten befassen sich mit zwei anderen Alzheimer assoziierten Proteinen, den Presenilinen. Bei den Presenilinen handelt es sich um Transmembranproteine, die die Membran 6 - 8 mal durchqueren. Sie besitzen bei familiären Alzheimerfällen eine große Bedeutung, da bestimmte Mutationen in den Presenilingenen zur Alzheimer'schen Krankheit führen. Dabei wurde gezeigt, daß im C. elegans Homologe zu den humanen Presenilinen (sel-12, spe-4, hop-1) existieren, wobei die Funktion der Preseniline in Mensch und Wurm konserviert ist ( Levitan D, Greenwald I (1995) Nature 377, 351 ; Levitan et al.(1996) Proc Natl Acad Sci USA, 93, 14940 ; Baumeister R (1997) Genes & Function 1, 149 ; Xiajun Li and Iva Greenwald (1997) Proc Natl Acad Sci USA, 94, 12204 ).C. elegans has already been used as a model organism in Alzheimer's disease, however, this work does not relate to the processing of APP into the Aβ peptide. Some papers deal with two other Alzheimer associated proteins, presenilins. The presenilins are transmembrane proteins that traverse the membrane 6-8 times. They are of great importance in familial Alzheimer cases, as certain mutations in the Presenilingen lead to Alzheimer's disease. It has been shown that C. elegans homologues to the human presenilines (sel-12, spe-4, hop-1) exist, wherein the function of presenilins is conserved in humans and worms ( Levitan D, Greenwald I (1995) Nature 377, 351 ; Levitan et al (1996) Proc Natl Acad Sci USA, 93, 14940 ; Baumeister R (1997) Genes & Function 1, 149 ; Xiajun Li and Iva Greenwald (1997) Proc Natl Acad Sci USA, 94, 12204 ).

Weitere Arbeiten beschäftigen sich mit dem APP-Homologen in C. elegans, das als Apl-1 bezeichnet wird und der Aß-Peptid Expression in C. elegans. Apl-1 besitzt jedoch keine Region, die zu der Aminosäuresequenz des Aß-Peptids homolog ist, weshalb C. elegans kein endogenes Aß-Peptid hat ( Daigle I, Li C (1993) Proc Natl Acad Sci USA, 90 (24), 12045 ).Further work is devoted to the APP homologue in C. elegans, designated Apl-1, and Aβ peptide expression in C. elegans. However, Apl-1 does not have a region homologous to the amino acid sequence of the Aβ peptide, therefore C. elegans does not have an endogenous Aβ peptide ( Daigle I, Li C (1993) Proc Natl Acad Sci USA, 90 (24), 12045 ).

Die Expression von Aß-Peptid (nicht die eines Aß-Vorläuferproteins) in C. elegans wurde von C. D. Link, Proc Natl Acad Sci USA (1995) 92, 9368 beschrieben. In diesen Arbeiten wurden transgene Würmer hergestellt, die ein Aß1-42 Peptid (d.h. das Aß-Peptid, das aus 42 Aminosäuren besteht) unter der Kontrolle des Muskelspezifischen Promotors unc 54 als Fusionsprotein mit einem synthetischen Signalpeptid exprimiert. In diesen Studien wurden Muskel-spezifische Proteinablagerungen, die mit anti-ß-amyloid Antikörpern reagierten, nachgewiesen. Weitere Arbeiten (z.B. C. Link et al. persönliche Mitteilung) beziehen sich auf Untersuchungen der Aggregation und Toxizität des Aß-Peptids im Modellsystem C. elegans.The expression of Aβ peptide (not that of an Aβ precursor protein) in C. elegans became by CD Link, Proc Natl Acad Sci USA (1995) 92, 9368 described. In this work, transgenic worms were produced expressing an Aβ1-42 peptide (ie the Aβ peptide consisting of 42 amino acids) under the control of the muscle specific promoter unc54 as a fusion protein with a synthetic signal peptide. In these studies, muscle-specific protein deposits that reacted with anti-β-amyloid antibodies were detected. Further work (eg, C. Link et al., Personal communication) relates to studies of the aggregation and toxicity of the Aβ peptide in the C. elegans model system.

Um die Existenz einer Prozessierungsmaschinerie in C. elegans, die an der Entstehung von Aß-Peptid beteiligt ist, zu untersuchen und potentielle Sekretasen im Wurm zu identifizieren wurden in der vorliegenden Arbeit transgene C. elegans Linien etabliert.In order to investigate the existence of a processing machinery in C. elegans, which is involved in the formation of Aß-peptide, and to identify potential secretases in the worm, transgenic C. elegans lines were established in the present work.

Gegenstand der vorliegenden Erfindung betrifft daher folgende Verwendung.The subject of the present invention therefore relates to the following use.

Verwendung eines transgenen C. elegans enthaltend ein Transgen enthaltend

  1. a) eine Nukleotidsequenz, die für das Amyloid Vorläufer Protein (APP) oder einen Teil davon kodiert,
    wobei die Nukleotidsequenz den Teil der Nukleotidsequenz des APPs umfasst, der für das vollständige Aβ-Peptid kodiert und
    wobei die Nukleotidsequenz nicht identisch ist mit dem Teil des APPs, der für ein vollständiges Aß-Peptid kodiert,
  2. b) gegebenenfalls ein oder mehrere weitere kodierende und/oder nicht-kodierende Nukleotidsequenzen und
  3. c) einen Promotor für die Expression in einer Zelle des Nematoden Caenorhabditis elegans (C. elegans)
    zur Identifizierung einer γ-Sekretase Aktivität und/oder einer α-Sekretase Aktivität in C. elegans.
Use of a transgenic C. elegans containing a transgene
  1. a) a nucleotide sequence coding for the amyloid precursor protein (APP) or a part thereof,
    wherein the nucleotide sequence comprises that part of the nucleotide sequence of the APP coding for the complete Aβ peptide and
    wherein the nucleotide sequence is not identical to the portion of the APP coding for a complete Aβ peptide,
  2. b) optionally one or more further coding and / or non-coding nucleotide sequences and
  3. c) a promoter for expression in a cell of the nematode Caenorhabditis elegans (C. elegans)
    to identify γ-secretase activity and / or α-secretase activity in C. elegans.

Vorzugsweise kodiert die Nukleotidsequenz für die 100 carboxy-terminalen Aminosäuren von APP, beginnend mit der Sequenz des Aß-Peptids und endend mit der carboxy-terminalen Aminosäure von APP, kodiert (C100 Fragment). Das APP ist vorzugsweise eines der Isoformen APP695 (695 Aminosäuren), APP751 (751 Aminosäuren), APP770 (770 Aminosäuren) und L-APP. Alle Isoformen entstehen durch alternatives Spleißen aus dem gleichen APP Gen. In APP695 wurden durch Spleißen die Exons 7 und 8 entfernt, wohingegen in APP751 nur das Exon 8 fehlt und in APP770 Exon 7 und 8 vorliegen. Daneben existieren weitere Spleißformen von APP, in denen das Exon 15 durch Spleißen entfernt wird. Diese Formen werden als L-APP bezeichnet und liegen ebenfalls in den bzgl. der Exons 7 und 8 gespleißten Formen vor.Preferably, the nucleotide sequence encodes the 100 carboxy-terminal amino acids of APP starting with the sequence of the Aβ peptide and ending with the carboxy-terminal amino acid of APP encoded (C100 fragment). The APP is preferably one of the isoforms APP695 (695 amino acids), APP751 (751 amino acids), APP770 (770 amino acids) and L-APP. All isoforms are formed by alternative splicing from the same APP gene. In APP695, exons 7 and 8 were removed by splicing, whereas in APP751 only exon 8 is absent and present in APP770 exons 7 and 8. In addition, there are other splice forms of APP, where the exon 15 is removed by splicing. These forms are referred to as L-APP and are also present in the forms spliced to exons 7 and 8.

In einer besonderen Ausführungsform der Erfindung enthält das Transgen die Nukleotidsequenz SEQ ID NO.: 1 oder einen Teil davon.In a particular embodiment of the invention, the transgene contains the nucleotide sequence SEQ ID NO .: 1 or a part thereof.

Vorzugsweise enthält das Transgen eine weitere kodierende Nukleotidsequenz, die am 5'-Ende der Nukleotidsequenz, die für APP oder einen Teil desselben kodiert, lokalisiert ist. In einer besonderen Ausführungsform der Erfindung kodiert die weitere Nukleotidsequenz für ein Signalpeptid oder einen Teil davon, beispielsweise für das APP Signalpeptid (SP) mit der Aminosäuresequenz SEQ ID NO.: 9 oder einen Teil davon.Preferably, the transgene contains a further coding nucleotide sequence located at the 5 'end of the nucleotide sequence encoding APP or a portion thereof. In a particular embodiment of the invention, the further nucleotide sequence codes for a signal peptide or a part thereof, for example for the APP signal peptide (SP) with the amino acid sequence SEQ ID NO .: 9 or a part thereof.

Die Sequenz vom N-Terminus des Aß-Peptids bis zum C-Terminus von APP besteht aus 99 Aminosäuren. Das APP-Signalpeptid besteht aus 17 Aminosäuren. Bei der Klonierung eines Fusionsproduktes bestehend aus dem N-Terminus des Aß-Peptids bis zum C-Terminus von APP und dem APP Signalpeptid, werden zwischen diese beiden Teile des Fusionsproduktes vorzugsweise ein oder mehrere "Spacer-Aminosäuren" eingefügt, vorzugsweise wird eine Aminosäure eingefügt, beispielsweise Leucin. Das C-Terminale Fragment wird deshalb unterschiedlich bezeichnet, z.B. C100 (C= C-Terminus), LC99 (L=Leucin), LC1-99, C99, SPA4CT (SP=Signalpeptid, A4=Aß-Peptid, CT= C-Terminus).The sequence from the N-terminus of the Aβ peptide to the C-terminus of APP consists of 99 amino acids. The APP signal peptide consists of 17 amino acids. When cloning a fusion product consisting of the N-terminus of the Aβ peptide to the C-terminus of APP and the APP signal peptide, one or more "spacer amino acids" are preferably inserted between these two parts of the fusion product, preferably an amino acid is inserted , for example leucine. The C-terminal fragment is therefore designated differently, e.g. C100 (C = C-terminus), LC99 (L = leucine), LC1-99, C99, SPA4CT (SP = signal peptide, A4 = Aβ-peptide, CT = C-terminus).

In einer besonderen Ausführungsfrom der Erfindung enthält das Transgen die Nukleotidsequenz SEQ ID NO.: 2 oder einen Teil davon und/oder die Nukleotidsequenz SEQ ID NO.: 3 oder eine Teil davon.In a particular embodiment of the invention, the transgene contains the nucleotide sequence SEQ ID NO .: 2 or a part thereof and / or the nucleotide sequence SEQ ID NO .: 3 or a part thereof.

Darüber hinaus kann das Transgen eine oder mehrere weitere nicht-kodierende und/oder eine oder mehrere weitere kodierende Nukleotidsequenzen enthalten. Beispielsweise kann das Transgen als weitere nicht-kodierende Nukleotidsequenz eine Sequenz aus einem Intron des APP Gens enthalten, z.B. eine Sequenz, die aus dem Intron 42 bp des APP Gens stammt und die Sequenz SEQ ID NO.: 4 aufweist. Gegenstand der Erfinndung ist ein Transgen, das die Nukleotidsequenz SEQ ID NO.: 5 enthält.In addition, the transgene may contain one or more other non-coding and / or one or more additional coding nucleotide sequences. For example, the transgene may contain, as a further non-coding nucleotide sequence, a sequence of an intron of the APP gene, for example a sequence which originates from the intron 42 bp of the APP gene and has the sequence SEQ ID NO .: 4. The object of the invention is a transgene which contains the nucleotide sequence SEQ ID NO .: 5.

Vorzugsweise enthält das Transgen auch ein oder mehrere genregulatorische Sequenzen für die geregelte Expression des kodierten Proteins, vorzugsweise einen konstitutiven oder einen regulierbaren Promotor. Beispielsweise kann der Promotor in neuronalem, muskulärem oder dermalem Gewebe von C. elegans aktive sein oder ubiquitär in C. elegans aktiv sein. Ein Promotor kann beispielsweise ausgewählt werden aus der Reihe der C. elegans Promotoren unc-54, hsp16-2, unc-119, goa-1 und sel-12. In einer besonderen Ausführungsform der Erfindung enthält das Transgen einen Promotor mit der Nukleotidsequenz SEQ ID NO.: 6. In einer besonderen Ausführungsform enthält das Transgen die Nukleotidsequenz SEQ ID NO.: 7.Preferably, the transgene also contains one or more gene regulatory sequences for the regulated expression of the encoded protein, preferably a constitutive or a regulatable promoter. For example, the promoter may be active in C. elegans neuronal, muscular or dermal tissue or ubiquitous in C. elegans. For example, a promoter may be selected from the range of C. elegans promoters unc-54, hsp16-2, unc-119, goa-1 and sel-12. In a particular embodiment of the invention, the transgene contains a promoter having the nucleotide sequence SEQ ID NO .: 6. In a particular embodiment, the transgene contains the nucleotide sequence SEQ ID NO .: 7.

Das Transgen kann in einem Vektor vorliegen, beispielsweise in einem Expressionsvektor. Beispielsweise kann ein rekombinanter Expressionsvektor die Nukleotidsequenz SEQ ID NO.: 8 enthalten.The transgene may be present in a vector, for example in an expression vector. For example, a recombinant expression vector may contain the nucleotide sequence of SEQ ID NO: 8.

Die Erfindung beschreibt auch die Herstellung eines Expressionsvektors, wobei ein Transgen nach bekannten Methoden in einen Vektor integriert wird. Insbesondere beschreibt die Erfindung die Verwendung eines Expressionsvektors zur Herstellung einer transgenen Zelle, wobei diese Zelle Teil eines nicht-humanen Organismus, z.B. C. elegans, sein kann.The invention also describes the production of an expression vector, wherein a transgene is integrated into a vector by known methods. In particular, the invention describes the use of an expression vector for the production of a transgenic cell, which cell is part of a non-human organism, e.g. C. elegans, can be.

Die Erfindung beschreibt auch die Herstellung des Transgens, wobei geeignete Teilsequenzen in der entsprechenden Reihenfolge und im richtigen Leserahmen, ggf. unter Einfügung von Linkern ligiert werden. Insbesondere beschreibt die Erfindung die Verwendung des Transgens, z.B. zur Herstellung einer transgenen Zelle, wobei diese Zelle Teil eines nicht humanen Organismus sein kann. Beispielsweise kann die Zelle eine C. elegans Zelle sein.The invention also describes the preparation of the transgene, wherein suitable partial sequences are ligated in the appropriate order and in the correct reading frame, if appropriate with the insertion of linkers. In particular, the invention describes the use of the transgene, eg for the production of a transgenic cell, which cell may be part of a non-human organism. For example, the cell may be a C. elegans cell.

Eine besondere Ausführungsform der Erfindung beschreibt einen transgenen C. elegans, der das Transgen enthält. Das Transgen kann in dem C. elegans auch in einem Expressionsvektor vorliegen. Das Transgen kann in dem C. elegans intra-und/oder extrachromosomal vorliegen. Intra- und/oder extrachromosomal können ein oder mehrere Transgene bzw. Expressionsvektoren, die das Transgen enthalten, alsA particular embodiment of the invention describes a transgenic C. elegans containing the transgene. The transgene may also be present in the C. elegans in an expression vector. The transgene may be present in the C. elegans intra- and / or extrachromosomal. Intra- and / or extrachromosomal, one or more transgenes or expression vectors containing the transgene, as

Long Tandem Arrays vorliegen. Vorzugsweise enthält eine transgene Zelle oder ein transgener Organismus zusätzlich einen weiteren Expressionsvektor, der eine Nukleotidsequenz enthält, die für einen Marker kodiert, wobei der Marker entweder ein temperatursensitiver oder ein phenotypischer Marker ist. Beispielsweise kann der Marker ein visueller oder ein verhaltens-phenotypischer Marker sein. Beispiele sind fluoreszierende Marker, z.B. GFP (green fluorescent protein) oder EGFP (Enhanced green fluorescent protein), Markergene, die für eine dominante mutierte Form eines bestimmten Proteins kodiert, z.B. für eine dominante RoI6 Mutation oder Markersequenzen, die für antisense RNA kodieren, z.B. für die antisense RNA von Unc-22 ist.Long tandem arrays are available. Preferably, a transgenic cell or transgenic organism additionally contains another expression vector containing a nucleotide sequence encoding a marker, which marker is either a temperature-sensitive or a phenotypic marker. For example, the marker may be a visual or a behavioral phenotypic marker. Examples are fluorescent labels, e.g. GFP (Green Fluorescent Protein) or EGFP (Enhanced Green Fluorescent Protein), marker genes encoding a dominant mutated form of a particular protein, e.g. for a dominant RoI6 mutation or marker sequences encoding antisense RNA, e.g. for the antisense RNA of Unc-22.

Vorzugsweise liegen in den Keimzellen und/oder den somatischen Zellen des transgenen C. elegans eine oder mehrere Kopien des Transgens und/oder des Expressionsvektors vor und ggf. eines weiteren Expressionsvektor vor.Preferably, one or more copies of the transgene and / or the expression vector are present in the germ cells and / or the somatic cells of the transgenic C. elegans and, if appropriate, a further expression vector.

Die Erfindung beschreibt auch Verfahren zur Herstellung eines transgenen C. elegans, wobei ein Transgen und/oder ein Expressionsvektor gegebenenfalls in Gegenwart eines weiteren Expressionsvektors, der eine Nukleotidsequenz enthält, die für einen Marker kodiert, in die Keimzellen eines C. elegans mikroinjiziert wird. Für die Herstellung der transgenen C. elegans-Linien kann beispielsweise ein DNA-Konstrukt verwendet, das SP-C100 (SP = Signalpeptid) unter der Kontrolle eines Neuron-spezifischen Promotors exprimiert (Abb. 2). Da sich C100 aus der Aβ-Sequenz und dem C-Terminus von APP zusammensetzt ist nur der γ-Sekretaseschnitt nötig, um das Aß-Peptid aus C100 freizusetzten. C100 stellt ebenfalls ein Substrat für die γ-Sekretase dar.The invention also describes methods for producing a transgenic C. elegans, wherein a transgene and / or an expression vector, if appropriate in the presence of a further expression vector which contains a nucleotide sequence which codes for a marker, is microinjected into the germinal cells of a C. elegans. For the production of the transgenic C. elegans lines, for example, a DNA construct expressing SP-C100 (SP = signal peptide) under the control of a neuron-specific promoter can be used ( Fig. 2 ). Since C100 is composed of the Aβ sequence and the C-terminus of APP, only the γ-secretase section is needed to liberate the Aβ peptide from C100. C100 also represents a substrate for γ-secretase.

Die Erfindung beschreibt auch die Verwendung eines transgenen C. elegans, beispielsweise zur Expression eines SP-C100 Fusionsproteins. Ein Gegenstand der Erfindung verwendet ein Sp-C100 Fusionsprotein mit der Aminosäuresequenz SEQ ID NO.: 10.The invention also describes the use of a transgenic C. elegans, for example for expression of an SP-C100 fusion protein. An object of the invention uses a Sp-C100 fusion protein having the amino acid sequence SEQ ID NO .: 10.

Insbesondere betrifft die Erfindung die Verwendung eines transgenen C. elegans zur Identifizierung einer γ-Sekretase Aktivität und/oder einer α-Sekretase Aktivität in C. elegans, die Verwendung in Verfahren zur Identifizierung und/oder Charakterisierung von Substanzen, die die γ-Sekretase Aktivität hemmen, die Verwendung in Verfahren zur Identifizierung und/oder Charakterisierung von Substanzen, die die α-Sekretase Aktivität erhöhen, die Verwendung in Verfahren zur Identifizierung und/oder Charakterisierung von Substanzen, die als Wirkstoffe zur Behandlung und/oder Prävention der Alzheimerschen' Krankheit verwendet werden können.In particular, the invention relates to the use of a transgenic C. elegans for the identification of γ-secretase activity and / or α-secretase activity in C. elegans, for use in methods for the identification and / or characterization of substances having the γ-secretase activity use in methods for the identification and / or characterization of substances that increase α-secretase activity, use in methods for the identification and / or characterization of substances used as active ingredients for the treatment and / or prevention of Alzheimer's disease can be.

Zur Identifizierung von Sekretasen, die an der Prozessierung von APP in das Aβ-Peptid beteiligt sind wurde in der vorliegenden Arbeit der Nematode Caenorhabditis elegans (C. elegans) als Modellorganismus gewählt. Dieser Wurm eignet sich hervorragend für genetische Studien und wurde daher in der Vergangenheit vielfach eingesetzt, um universell wichtige Prozesse wie z.B. den programmierten Zelltod, Neuronal guidance und RAS/MAP Kinase Signalling zu untersuchen (Riddle, D.L. et al. (1997)).To identify secretases involved in the processing of APP into the Aβ peptide, the nematode Caenorhabditis elegans (C. elegans) was chosen as the model organism in the present work. This worm is excellently suited for genetic studies and has therefore been widely used in the past to detect universally important processes, e.g. to study programmed cell death, neuronal guidance, and RAS / MAP kinase signaling (Riddle, D.L. et al., 1997).

Zu den wesentlichen Punkten, durch die sich C. elegans besonders für solche Studien auszeichnet, zählen ( C. Kenyon, Science (1988) 240, 1448 ; P.E. Kuwabara (1997), TIG, 13, 454 ):

  • sein kleines Genom, das sich aus etwa 19000 Genen bzw. 97 Mb zusammensetzt und im Dezember 1998 vollständig sequenziert worden ist. (The C. elegans Sequencing Consortium, Science (1998), 282, 2012).
  • seine Vermehrung durch Selbstbefruchtung. Bei den zwei Geschlechtern von C. elegans unterscheidet man zwischen Männchen und Hermaphroditen, d.h. zwittrige Tiere, die ihre Eier vor der Ablage selbst befruchten. Ein entscheidender Vorteil dieser Art der Vermehrung ist, daß ein Hermaphrodit nach dem Einbringen eines Transgens in die Keimbahn automatisch homozygote transgene Nachkommen erzeugen kann. Es sind also keine weiteren Kreuzungsschritte wie z.B. bei Drosophila zur Herstellung transgener Linien notwendig.
  • seine leichte Handhabung im Labor aufgrund seiner geringen Größe (etwa 1 mm Länge) und seiner relativ anspruchslosen Wachstumsbedingungen. Es kann daher eine große Zahl von Würmern routinemäßig im Labor gehandhabt werden.
  • seine kurze Generationszeit von 3 Tagen, die es ermöglicht innerhalb sehr kurzer Zeit große Mengen an biologischem Material für Analysen zu erhalten.
  • Es ist eine komplette Zellbeschreibung für die Entwicklung und Anatomie von C. elegans vorhanden.
  • Es liegen detaillierte genetische Karten und Methoden zur genetischen Analyse in C. elegans vor.
  • Es sind Technologien zur Herstellung von knock-out Tieren vorhanden. Ebenso existieren Technologien zur Mutagenisierung des C. elegans Genoms (Transposon Mutagenese, Ethylmethansulfonate (EMS) Mutagenese).
Among the key points that distinguish C. elegans especially for such studies are ( C. Kenyon, Science (1988) 240, 1448 ; PE Kuwabara (1997), TIG, 13, 454 ):
  • its small genome, which consists of about 19,000 genes or 97 Mb and was fully sequenced in December 1998. (The C. elegans Sequencing Consortium, Science (1998), 282, 2012).
  • its multiplication by self-fertilization. In the two sexes of C. elegans a distinction is made between males and hermaphrodites, ie hermaphroditic animals that self-fertilize their eggs before storing. A decisive advantage of this type of propagation is that a hermaphrodite can automatically generate homozygous transgenic offspring after introduction of a transgene into the germline. Thus, there are no further crossing steps such as Drosophila necessary for the production of transgenic lines.
  • its ease of use in the laboratory due to its small size (about 1 mm in length) and its relatively undemanding growth conditions. Therefore, a large number of worms can be routinely handled in the laboratory.
  • its short generation time of 3 days, which makes it possible to obtain large quantities of biological material for analysis in a very short time.
  • There is a complete cell description available for the development and anatomy of C. elegans.
  • There are detailed genetic maps and methods for genetic analysis in C. elegans.
  • There are technologies for producing knock-out animals. Also, there are technologies for mutagenizing the C. elegans genome (transposon mutagenesis, ethylmethane sulfonate (EMS) mutagenesis).

Verwendungsmöglichkeiten der transgenen C. elegans Linien:

  1. 1. Identifizierung einer γ-Sekretase ähnlichen Aktivität in C. elegans mit Hilfe von Mutageneseansätzen. Geplant ist eine Transposonmutagenese, durch die die γ-Sekretase ähnliche Aktivität zerstört und durch Detektion der Würmer, die diese Aktivität nicht mehr besitzen, das entsprechende Gen gesucht werden soll. Solch ein Screeningverfahren ist in der Literatur beschrieben: Korswagen H.C. et al., (1996), 93, 14680 Proc Natl Acad Sci USA ).
    Alternative Ansätze wären Mutagenese über Ethylmethansulfonat (EMS) oder auch RNA anti-sense Ansätze. Bei letzterem könnte man versuchen gemeinsame Motive aller C. elegans Proteasen zu finden und gezielt mit anti-sense RNA, die gegen diese Motive gerichtet sind, herunter zu regulieren. Ein Screening auf das Aß-Peptid könnte dann zeigen, ob eine der Proteasen an der Aß-Produktion beteiligt ist.
  2. 2. Identifizierung einer γ-Sekretase ähnlichen Aktivität in C. elegans evtl. über einen ähnlichen Weg wie in Punkt 1. beschrieben.
  3. 3. Mit der Kenntnis einer γ-Sekretase bzw. γ-Sekretase ähnlichen Aktivität in C. elegans kann über Homologievergleich die humane γ-Sekretase bzw. γ-Sekretase ähnliche Aktivität gesucht werden.
  4. 4. Identifizierung von Pharmaka, die
    • die γ-Sekretaseaktivität hemmen, um direkt die Aß-Produktion aus dem Amyloidvorläuferprotein zu inhibieren.
    • die γ-Sekretase aktivieren und dadurch indirekt durch eine Steigerung der APPsec-Produktion die Entstehung des Aß-Peptids inhibieren.
      Dieser Ansatz könnte im 96-Well-Format stattfinden, da C. elegans in Suspension in 96-Well-Platten gehalten werden kann.
      Da das Screening an einem Gesamtorganismus durchgeführt wird, können Pharmaka mit unspezifisch toxischer Wirkung weitgehend ausgeschlossen werden.
  5. 5. Untersuchung des Aggregationsverhaltens und einer eventuellen neurotoxischen Wirkung des Aß-Peptids in C. elegans. Screening nach Pharmaka, die die Aggregation von Aß-Peptid inhibieren.
  6. 6. Untersuchung zur Modulation der APP-Prozessierung durch andere Proteine (z.B. Preseniline oder ApoE) durch deren Überexpression oder knock-out. Da es sich bei den Presenilinen um Alzheimer-assoziierte Proteine handelt und ApoE ein Risikofaktor der Alzheimer Krankheit darstellt könnten diese Proteine die Bildung des Aß-Peptids beeinflussen und damit ihre Rolle im APP-Prozessierungsweg untersucht werden.
  7. 7. Gegebenenfalls Validierung einer α- und/oder γ-Sekretaseaktivität, die mit Hilfe anderer, dem Fachmann bekannter, experimenteller Ansätze gefunden wurde.
Uses of transgenic C. elegans lines:
  1. 1. Identification of a γ-secretase-like activity in C. elegans using mutagenesis approaches. A transposon mutagenesis is planned, by which the γ-secretase-like activity is destroyed and by detection of the worms, which no longer possess this activity, the corresponding gene should be sought. Such a screening method is described in the literature: Korswagen HC et al., (1996), 93, 14680 Proc Natl Acad Sci USA ).
    Alternative approaches would be mutagenesis via ethyl methanesulfonate (EMS) or RNA anti-sense approaches. In the latter case, one could try to find common motifs of all C. elegans proteases and downregulate them specifically with antisense RNA directed against these motifs. Screening for the Aβ peptide could then show if any of the proteases are involved in Aβ production.
  2. 2. Identification of γ-secretase-like activity in C. elegans, possibly via a similar route as described in point 1.
  3. 3. With knowledge of γ-secretase or γ-secretase-like activity in C. elegans, homology comparison can be used to search for human γ-secretase or γ-secretase-like activity.
  4. 4. Identification of pharmaceuticals that
    • inhibit γ-secretase activity to directly inhibit Aβ production from the amyloid precursor protein.
    • activate the γ-secretase and thereby indirectly inhibit the production of the Aβ peptide by increasing the production of APPsec.
      This approach could take place in 96 well format since C. elegans can be kept in suspension in 96 well plates.
      Since the screening is performed on an entire organism, drugs with unspecific toxic effects can be largely excluded.
  5. 5. Investigation of the aggregation behavior and a possible neurotoxic effect of the A.beta. Peptide in C. elegans. Screening for drugs that inhibit the aggregation of Aβ peptide.
  6. 6. Investigation of modulation of APP processing by other proteins (eg, presenilins or ApoE) by their overexpression or knock-out. Since presenilins are Alzheimer-associated proteins and ApoE represents a risk factor for Alzheimer's disease, these proteins could influence the formation of the Aβ peptide and thus their role in the APP processing pathway.
  7. 7. If applicable, validation of α- and / or γ-secretase activity found by other experimental approaches known to those skilled in the art.

Figur 1: Figur 1 zeigt das Amyloid Vorläuferprotein (Isoform APP695 und Isoformen APP770 bzw. APP751) und Sekretase Spaltprodukte. FIG. 1: FIG. 1 shows the amyloid precursor protein (isoform APP695 and isoforms APP770 and APP751, respectively) and secretase cleavage products.

Figur 2: Figur 2 beschreibt die Konstruktion des transgenen Vektors "Unc-119-SP-C100" enthaltend einem unc-119 Promotor, ein APP-Signalpeptid und das C100 Fragment aus APP, wobei "unc-119" ein Neuron-spezifischer C. elegans Promotor ist, APP Signalpeptid, den Aminosäuren 1 bis 24 von APP entspricht und C100 den C-terminalen 100 Aminosäuren von APP (= C100) entspricht. C100 besteht aus der Aß-Sequenz und dem C-Terminus von APP ( Shoji, M et al., (1992) Science 258, 126 ). Der Vektor Unc-119-SP-C100 hat 5112 Basenpaare. FIG. 2: FIG. 2 describes the construction of the transgenic vector "Unc-119-SP-C100" containing an unc-119 promoter, an APP signal peptide and the C100 fragment from APP, where "unc-119" is a neuron-specific C. elegans promoter, APP Signal peptide corresponding to amino acids 1 to 24 of APP and C100 corresponds to the C-terminal 100 amino acids of APP (= C100). C100 consists of the Aβ sequence and the C-terminus of APP ( Shoji, M et al., (1992) Science 258, 126 ). The vector Unc-119-SP-C100 has 5112 base pairs.

Beispiele:Examples: Beipiel 1: Herstellung eines Expressionsvektors, der das Transgen enthält.Example 1: Preparation of an expression vector containing the transgene.

Für die Klonierung wurden zwei Vektoren verwendet, pSKLC1-99, der das SP-C100 kodiert und pBY103, der den unc-119 Promotor enthält, wobei die für SP-C100 kodierende DNA hinter den unc-119 Promotor in den pBY103-Vektor kloniert wurde. Der Basisvektor pBY103 besteht aus dem Vektorbackbone pPD49.26, beschrieben in " Caenorhabditis elegans: Modern Biological Analysis of an Organism" (1995) Ed. Epstein et al., Vol 48, pp. 473 in das der unc-119 Promotor ( Maduro et al. Genetics (1995), 141, p. 977 ) über HindIII/BamHI kloniert wurde. Das Plasmid unc-119-SP-C100 wurde durch KpnI/SacI Schnitt und anschließende Klonierung des LC99 Fragments aus pSKLC1-99 (Shoji et al. (1992) in den pBY103 hergestellt.Two vectors were used for the cloning, pSKLC1-99, which encodes the SP-C100 and pBY103, which contains the unc-119 promoter, wherein the DNA coding for SP-C100 was cloned into the pBY103 vector behind the unc-119 promoter , The base vector pBY103 consists of the vector backbone pPD49.26 described in " Caenorhabditis elegans: Modern Biological Analysis of an Organism "(1995) Ed. Epstein et al., Vol 48, pp. 473 into which the unc-119 promoter ( Maduro et al. Genetics (1995), 141, p. 977 ) was cloned via HindIII / BamHI. The plasmid unc-119-SP-C100 was prepared by KpnI / SacI cut and subsequent cloning of the LC99 fragment from pSKLC1-99 (Shoji et al. (1992) into the pBY103.

Beispiel 2: Herstellung der transgenen C. elegans LinienExample 2: Preparation of transgenic C. elegans lines

Zur Herstellung der transgenen C. elegans Linien wurde das Verfahren der Mikroinjektion angewandt ( Mello et al.,(1991) EMBO J. 10 (12) 3959 ; C. Mello and A. Fire, Methods in Cell Biology, Academic Press Vol48, pp451, 1995 ; C. D. Link, Proc Natl Acad Sci USA (1995) 92, 9368 ).For the preparation of the transgenic C. elegans lines, the method of microinjection was used ( Mello et al., (1991) EMBO J. 10 (12) 3959 ; C. Mello and A. Fire, Methods in Cell Biology, Academic Press Vol. 48, pp451, 1995 ; CD Link, Proc Natl Acad Sci USA (1995) 92, 9368 ).

Verwendet wurden zwei verschiedene C. elegans-Stämme, Wild-typ N2 und him-8 (high incidence of males). Das unc-119-SP-C100-Konstrukt wurde in die Gonaden von jungen adulten Hermaphroditen mit Hilfe einer Mikroinjektionsanlage mikroinjiziert. Die DNA-Konzentration betrug etwa 20 ng/µl.Two different C. elegans strains were used, wild type N2 and him-8 (high incidence of males). The unc-119 SP-C100 construct was microinjected into the gonads of young adult hermaphrodites using a microinjection system. The DNA concentration was about 20 ng / μl.

Zusammen mit dem unc-119-SP-C100 wurde ein Markerplasmid injiziert. Dabei handelt es sich um das ttx3-GFP-Plasmid, das für das Green Fluorescent Protein unter der Kontrolle des ttx3-Promotors kodiert. Die Aktivität des ttx3-Promotors ist spezifisch für bestimmte Kopfneuronen von C. elegans, den sogenannten AIY-Neuronen, die bei der Thermotaxis des Wurms eine Rolle spielen.Together with the unc-119-SP-C100, a marker plasmid was injected. It is the ttx3-GFP plasmid that encodes the Green Fluorescent Protein under the control of the ttx3 promoter. The activity of the ttx3 promoter is specific for certain head neurons of C. elegans, the so-called AIY neurons, which play a role in the thermotaxis of the worm.

Bei der Mikroinjektion von Plasmid-DNA wird davon ausgegangen, daß sich durch Rekombination sogenannte Long Tandem Arrays bilden, die aus vielen Kopien von Plasmid-DNA (in unserem Falle aus dem ttx3-GFP und dem unc-119-SP-C100) bestehen. Diese Arrays integrieren zu einem bestimmten Prozentsatz in das C. elegans Genom. Mit einer höheren Wahrscheinlichkeit jedoch liegen die Arrays extrachromosomal vor.In the microinjection of plasmid DNA, recombination is thought to produce so-called long tandem arrays consisting of many copies of plasmid DNA (in our case, the ttx3-GFP and the unc-119-SP-C100). These arrays integrate into the C. elegans genome to a certain percentage. With a higher probability, however, the arrays are extrachromosomal.

Erfolgreich injizierte Würmer weisen bei Anregung mit Licht einer Wellenlänge von etwa 480 nm grüne Fluoreszenz in den AIY-Neuronen im Kopfbereich auf. Solche Nematoden konnten detektiert werden.Successfully injected worms exhibit green fluorescence in the head of the AIY neurons when excited with light at a wavelength of about 480 nm. Such nematodes could be detected.

Beispiel 3: Beschreibung der C100 transgenen C. elegans LinienExample 3: Description of the C100 Transgenic C. elegans Lines 1. Phänotypische Merkmale1. Phenotypic features

C100 transgene Würmer weisen in den AIY-Neuronen des Kopfbereiches nach Anregung mit Licht der Wellenlänge von 480 nm eine grüne Fluoreszenz auf. Da auch bei den Nachkommen der Würmer wiederum grüne Fluoreszenz in den Kopfneuronen detektierbar war, kann davon ausgegangen werden, daß die Plasmide keimbahngängig sind. Jedoch ist die Penetranz nicht 100 %, was darauf schließen läßt, daß die long tandam arrays aus ttx3-GFP-Marker-DNA und unc-119-SP-C100 eher extrachromosomal als in das Genom integriert vorliegen.C100 transgenic worms show a green fluorescence in the AIY neurons of the head area after excitation with light of the wavelength of 480 nm. Since green fluorescence was again detectable in the head neurons in the offspring of the worms, it can be assumed that the plasmids are pathway-friendly. However, the penetrance is not 100%, suggesting that the long tandam arrays of ttx3-GFP marker DNA and unc-119-SP-C100 are more extrachromosomally integrated than in the genome.

Beispiel 4: Nachweis der C100 Expression im BlotExample 4: Detection of C100 Expression in the Blot

Sechs verschiedene transgene C100 C. elegans Linien (drei im N2 wt und drei im him 8 Hintergrund) wurden in einem Western Blot auf die Expression des C100 Fragments mit einem polyklonalen Antiserum, das gegen den C-Terminus von APP gerichtet ist, untersucht. In allen sechs Linien war eine Bande mit dem entsprechenden Molekulargewicht von etwa 10 kDa detektierbar.Six different transgenic C100 C. elegans lines (three in the N2 wt and three in the background 8 background) were examined in a Western blot for expression of the C100 fragment with a polyclonal antiserum directed against the C-terminus of APP. In all six lines, a band with the corresponding molecular weight of about 10 kDa was detectable.

Beispiel 5: Nachweis des C100 im ELISAExample 5: Detection of the C100 in the ELISA

In einem Aβ Sandwich ELISA konnten in Zellextrakten aus transgenen Tieren Signale über dem Hintergrund-Level detektiert werden, die in zwei Fällen statistisch signifikant waren. Dies deutet darauf hin, daß C. elegans eine γ-Sekretase ähnliche Aktivität besitzen könnte.In an Aβ sandwich ELISA, cell extracts from transgenic animals were able to detect signals above the background level, which were statistically significant in two cases. This indicates that C. elegans might have a γ-secretase-like activity.

Bei dem Aß-Sandwich ELISA Assay werden 96-Well-Platten zunächst mit dem monoklonalen Antikörper Klon 6E10 (SENETEK PLC., MO, USA), der spezifisch mit dem Aß-Peptid (Aminosäure 1-17) reagiert, inkubiert und anschließend mit Wurmextrakten aus transgenen Würmern bzw. Kontrollwürmern überschichtet. Die Detektion des Aß-Peptids findet mit Hilfe des monoklonalen Aß-Antikörper 4G8 (SENETEK PLC., MO, USA), der die Aminosäuren 17-24 im Aß-Peptid erkennt und mit Biotin markiert ist, statt. Der Nachweis erfolgt über die Alkalische Phosphatase Reaktion mit einem entsprechenden Antikörper der gegen Biotin gerichtet ist. Der Aufschluß der Würmer impliziert Detergenzbehandlung, Stickstoffschockgefrieren, Sonifizierung, und das Aufbrechen der Zellen mit Glasperlen.In the Aβ sandwich ELISA assay, 96-well plates are first incubated with the monoclonal antibody clone 6E10 (SENETEK PLC., MO, USA), which reacts specifically with the Aβ peptide (amino acid 1-17), and then with worm extracts from transgenic worms or control worms. The detection of the Aβ peptide takes place with the aid of the monoclonal Aβ antibody 4G8 (SENETEK PLC., MO, USA), which recognizes amino acids 17-24 in the Aβ peptide and is labeled with biotin. The detection is carried out via the alkaline phosphatase reaction with a corresponding antibody which is directed against biotin. The digestion of the worms implies detergent treatment, nitrogen shock freezing, sonication, and rupture of the cells with glass beads.

Das ELISA Signal aus dem oben beschriebenen Versuch kann sowohl einer schwachen Expression des Aß-Peptids als auch der des C100-Vorläuferproteins zugrunde liegen, da die entsprechenden Epitope in beiden Proteinen vorliegen.The ELISA signal from the experiment described above may underlie both weak expression of the Aβ peptide and that of the C100 precursor protein, since the corresponding epitopes are present in both proteins.

In analoger Weise könnte beispielsweise auch die Expression des Aß-Peptides spezifisch nachgewiesen werden: Dazu müßten Aß-spezifische Antikörper, die nicht mit dem C 100-Vorläufer reagieren, in einem Aß-Sandwich ELISA eingesetzt werden. Ein Aß-spezifischer Antikörper könnte beispielsweise ein monoklonaler Antikörper sein, der spezifisch das C-terminale Ende der Aß-Form erkennt, die sich aus 40 oder aus 42 Aminosäuren zusammensetzt. Parallel könnte das Aß-Peptid im Western Blot mit Hilfe der monoklonalen Antikörper 4G8 und 6E10 detektiert und dann aufgrund seines Molekulargewichtes von 4kD von dem größeren C100-Vorläufer unterschieden werden.In an analogous manner, for example, the expression of the Aβ peptide could be detected specifically: For this purpose, Aß-specific antibodies that do not react with the C 100 precursor would have to be used in an Aβ sandwich ELISA. For example, an Aβ-specific antibody could be a monoclonal antibody that specifically recognizes the C-terminal end of the Aβ form, which is composed of 40 or 42 amino acids. In parallel, the Aβ peptide could be detected in the Western blot using monoclonal antibodies 4G8 and 6E10 and then discriminated from the larger C100 precursor due to its molecular weight of 4kD.

Die Vektoren sind erhältlich bei Andrew Fire (Department of Embryology, Carnegie Institution of Washington, Baltimore, Maryland 21210, USA) für den pPD49.26 bzw. LC99 (Amyloid Precursor protein) hinterlegt unter der ATCC-Nummer 106372. Der unc-119-Promotor ist erhältlich von Maduro, M. (Department of Biological Science, Universitiy of Alberta Edmonton, Canada), unc-54 und unc-16.2 sind von Andrew Fire erhältlich.

  • SEQ ID NO.1: Nukleotidsequenz von C100
    Figure imgb0001
  • SEQ ID NO.2: Nukleotidsequenz von SP
    ATG CTGCCCGGTT TGGCACTGTT CCTGCTGGCC GCCTGGACGG CTCGGGCG
  • SEQ ID NO.3: Nukleotidsequenz von SP+C100
    Figure imgb0002
  • SEQ ID NO.4: Nukleotidsequenz Intron 42bp
    GTATGTTTCGAATGATACTAACATAACATAGAACATTTTCAG
  • SEQ ID NO.5: Nukleotidsequenz von Intron+SP+C100
    Figure imgb0003
  • SEQ ID NO.6: Nukleotidsequenz von unc-119
    Figure imgb0004
    Figure imgb0005
  • SEQ ID NO.7: Nukleotidsequenz von unc-119+ Intron+SP+C100
    Figure imgb0006
    Figure imgb0007
  • SEQ ID NO.8: Nukleotidsequenz des Expressionsvektors
    Figure imgb0008
    Figure imgb0009
  • SEQ ID NO.9: Aminosäuresequenz von SP
    MLPGLALFLL AAWTARA
  • SEQ ID NO.10: Aminosäuresequenz des Fusionsproteins
    MLPGLALFLL AAWTARALDA EFRHDSGYEV HHQKLVFFAE DVGSNKGAII
    GLMVGGVVIA TVIVITLVML KKKQYTSIHH G\/VEVDAAVT PEERHLSKMQ
    QNGYENPTYK FFEQMQN
  • SEQ ID NO. 11: Nukleotidsequenz des Vektors unc-119-SP-C100
    Figure imgb0010
    Figure imgb0011
    Figure imgb0012
The vectors are available from Andrew Fire (Department of Embryology, Carnegie Institution of Washington, Baltimore, Md. 21210, USA) for the pPD49.26 and / or amyloid precursor protein (LC99) deposited under the ATCC number 106372. The unc-119- Promoter is available from Maduro, M. (Department of Biological Science, University of Alberta Edmonton, Canada), unc-54 and unc-16.2 are available from Andrew Fire.
  • SEQ ID NO.1: nucleotide sequence of C100
    Figure imgb0001
  • SEQ ID NO.2: Nucleotide sequence of SP
    ATG CTGCCCGGTT TGGCACTGTT CCTGCTGGCC GCCTGGACGG CTCGGGCG
  • SEQ ID NO.3: Nucleotide sequence of SP + C100
    Figure imgb0002
  • SEQ ID NO.4: nucleotide sequence intron 42bp
    GTATGTTTCGAATGATACTAACATAACATAGAACATTTTCAG
  • SEQ ID NO. 5: Nucleotide sequence of intron + SP + C100
    Figure imgb0003
  • SEQ ID NO.6: nucleotide sequence of unc-119
    Figure imgb0004
    Figure imgb0005
  • SEQ ID NO.7: nucleotide sequence of unc-119 + intron + SP + C100
    Figure imgb0006
    Figure imgb0007
  • SEQ ID NO.8: Nucleotide sequence of the expression vector
    Figure imgb0008
    Figure imgb0009
  • SEQ ID NO.9: Amino acid sequence of SP
    MLPGLALFLL AAWTARA
  • SEQ ID NO.10: Amino acid sequence of the fusion protein
    MLPGLALFLL AAWTARALDA EFRHDSGYEV HHQKLVFFAE DVGSNKGAII
    GLMVGGVVIA TVIVITLVML KKKQYTSIHH G \ / VEVDAAVT PEERHLSKMQ
    QNGYENPTYK FFEQMQN
  • SEQ ID NO. Figure 11: Nucleotide sequence of vector unc-119-SP-C100
    Figure imgb0010
    Figure imgb0011
    Figure imgb0012

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      2. (B) LOCATION: 1..354 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
        Figure imgb0014
  • (2) ANGABEN ZU SEQ ID NO: 4:
    • (i) SEQUENZKENNZEICHEN:
      1. (A) LÄNGE: 42 Basenpaare
      2. (B) ART: Nucleotid
      3. (C) STRANGFORM: Einzelstrang
      4. (D) TOPOLOGIE: linear
    • (ii) ART DES MOLEKÜLS: Genom-DNA (ix) MERKMAL:
      1. (A) NAME/SCHLÜSSEL: exon
      2. (B) LAGE:1..42
    • (xi) SEQUENZBESCHREIBUNG: SEQ ID NO: 4:
      GTATGTTTCG AATGATACTA ACATAACATA GAACATTTTC AG    42
    (2) PARTICULARS TO SEQ ID NO: 4:
    • (i) SEQUENCE MARKINGS:
      1. (A) LENGTH: 42 base pairs
      2. (B) ART: nucleotide
      3. (C) STRING FORM: single strand
      4. (D) TOPOLOGY: linear
    • (ii) ART OF MOLECULAR: Genomic DNA (ix) FEATURE:
      1. (A) NAME / KEY: exon
      2. (B) LOCATION: 1..42
    • (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
      GTATGTTTCG AATGATACTA ACATAACATA GAACATTTTC AG 42
  • (2) ANGABEN ZU SEQ ID NO: 5:
    • (i) SEQUENZKENNZEICHEN:
      1. (A) LÄNGE: 495 Basenpaare
      2. (B) ART: Nucleotid
      3. (C) STRANGFORM: Einzelstrang
      4. (D) TOPOLOGIE: linear
    • (ii) ART DES MOLEKÜLS: Genom-DNA
    • (ix) MERKMAL:
      1. (A) NAME/SCHLÜSSEL: exon
      2. (B) LAGE:1..495 (xi) SEQUENZBESCHREIBUNG: SEQ ID NO: 5:
        Figure imgb0015
    (2) PARTICULARS TO SEQ ID NO: 5:
    • (i) SEQUENCE MARKINGS:
      1. (A) LENGTH: 495 base pairs
      2. (B) ART: nucleotide
      3. (C) STRING FORM: single strand
      4. (D) TOPOLOGY: linear
    • (ii) ART OF MOLECULAR: Genomic DNA
    • (ix) FEATURE:
      1. (A) NAME / KEY: exon
      2. (B) LOCATION: 1..495 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
        Figure imgb0015
  • (2) ANGABEN ZU SEQ ID NO: 6:
    • (i) SEQUENZKENNZEICHEN:
      1. (A) LÄNGE: 1207 Basenpaare
      2. (B) ART: Nucleotid
      3. (C) STRANGFORM: Einzelstrang
      4. (D) TOPOLOGIE: linear
    • (ii) ART DES MOLEKÜLS: Genom-DNA
    • (ix) MERKMAL:
      1. (A) NAME/SCHLÜSSEL: exon
      2. (B) LAGE:1..1207
    • (xi) SEQUENZBESCHREIBUNG: SEQ ID NO: 6:
      Figure imgb0016
    (2) PARTICULARS TO SEQ ID NO: 6:
    • (i) SEQUENCE MARKINGS:
      1. (A) LENGTH: 1207 base pairs
      2. (B) ART: nucleotide
      3. (C) STRING FORM: single strand
      4. (D) TOPOLOGY: linear
    • (ii) ART OF MOLECULAR: Genomic DNA
    • (ix) FEATURE:
      1. (A) NAME / KEY: exon
      2. (B) LOCATION: 1..1207
    • (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
      Figure imgb0016
  • (2) ANGABEN ZU SEQ ID NO: 7:
    • (i) SEQUENZKENNZEICHEN:
      1. (A) LÄNGE: 1773 Basenpaare
      2. (B) ART: Nucleotid
      3. (C) STRANGFORM: Einzelstrang
      4. (D) TOPOLOGIE: linear
    • (ii) ART DES MOLEKÜLS: Genom-DNA
    • (ix) MERKMAL:
      1. (A) NAME/SCHLÜSSEL: exon
      2. (B) LAGE:1..1773
    • (xi) SEQUENZBESCHREIBUNG: SEQ ID NO: 7:
      Figure imgb0017
      Figure imgb0018
    (2) PARTICULARS TO SEQ ID NO: 7:
    • (i) SEQUENCE MARKINGS:
      1. (A) LENGTH: 1773 base pairs
      2. (B) ART: nucleotide
      3. (C) STRING FORM: single strand
      4. (D) TOPOLOGY: linear
    • (ii) ART OF MOLECULAR: Genomic DNA
    • (ix) FEATURE:
      1. (A) NAME / KEY: exon
      2. (B) LOCATION: 1..1773
    • (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:
      Figure imgb0017
      Figure imgb0018
  • (2) ANGABEN ZU SEQ ID NO: 8:
    • (i) SEQUENZKENNZEICHEN:
      1. (A) LÄNGE: 3344 Basenpaare
      2. (B) ART: Nucleotid
      3. (C) STRANGFORM: Einzelstrang
      4. (D) TOPOLOGIE: linear
    • (ii) ART DES MOLEKÜLS: Genom-DNA
    • (ix) MERKMAL:
      1. (A) NAME/SCHLÜSSEL: exon
      2. (B) LAGE:1..3344
    • (xi) SEQUENZBESCHREIBUNG: SEQ ID NO: 8:
      Figure imgb0019
      Figure imgb0020
      Figure imgb0021
    (2) PARTICULARS TO SEQ ID NO: 8:
    • (i) SEQUENCE MARKINGS:
      1. (A) LENGTH: 3344 base pairs
      2. (B) ART: nucleotide
      3. (C) STRING FORM: single strand
      4. (D) TOPOLOGY: linear
    • (ii) ART OF MOLECULAR: Genomic DNA
    • (ix) FEATURE:
      1. (A) NAME / KEY: exon
      2. (B) LOCATION: 1..3344
    • (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:
      Figure imgb0019
      Figure imgb0020
      Figure imgb0021
  • (2) ANGABEN ZU SEQ ID NO: 9:
    • (i) SEQUENZKENNZEICHEN:
      1. (A) LÄNGE: 17 Aminosäuren
      2. (B) ART: Aminosäure
      3. (C) STRANGFORM: Einzelstrang
      4. (D) TOPOLOGIE: linear
    • (ii) ART DES MOLEKÜLS: Protein
    • (ix) MERKMAL:
      1. (A) NAME/SCHLÜSSEL: Protein
      2. (B) LAGE:1..17
    • (xi) SEQUENZBESCHREIBUNG: SEQ ID NO: 9:
      Figure imgb0022
    (2) PARTICULARS TO SEQ ID NO: 9:
    • (i) SEQUENCE MARKINGS:
      1. (A) LENGTH: 17 amino acids
      2. (B) ART: amino acid
      3. (C) STRING FORM: single strand
      4. (D) TOPOLOGY: linear
    • (ii) ART OF MOLECULAR: Protein
    • (ix) FEATURE:
      1. (A) NAME / KEY: Protein
      2. (B) LOCATION: 1..17
    • (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:
      Figure imgb0022
  • (2) ANGABEN ZU SEQ ID NO: 10:
    • (i) SEQUENZKENNZEICHEN:
      1. (A) LÄNGE: 117 Aminosäuren
      2. (B) ART: Aminosäure
      3. (C) STRANGFORM: Einzelstrang
      4. (D) TOPOLOGIE: linear
    • (ii) ART DES MOLEKÜLS: Protein
    • (ix) MERKMAL:
      1. (A) NAME/SCHLÜSSEL: Protein
      2. (B) LAGE:1..117 (xi) SEQUENZBESCHREIBUNG: SEQ ID NO: 10:
        Figure imgb0023
    (2) PARTICULARS TO SEQ ID NO: 10:
    • (i) SEQUENCE MARKINGS:
      1. (A) LENGTH: 117 amino acids
      2. (B) ART: amino acid
      3. (C) STRING FORM: single strand
      4. (D) TOPOLOGY: linear
    • (ii) ART OF MOLECULAR: Protein
    • (ix) FEATURE:
      1. (A) NAME / KEY: Protein
      2. (B) LOCATION: 1..117 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:
        Figure imgb0023
  • (2) ANGABEN ZU SEQ ID NO: 11:
    • (i) SEQUENZKENNZEICHEN:
      1. (A) LÄNGE: 5109 Basenpaare
      2. (B) ART: Nucleotid
      3. (C) STRANGFORM: Einzelstrang
      4. (D) TOPOLOGIE: linear
    • (ii) ART DES MOLEKÜLS: Genom-DNA
    • (ix) MERKMAL:
      1. (A) NAME/SCHLÜSSEL: exon
      2. (B) LAGE:1..5109
    • (xi) SEQUENZBESCHREIBUNG: SEQ ID NO: 11:
      Figure imgb0024
      Figure imgb0025
      Figure imgb0026
      Figure imgb0027
    (2) PARTICULARS TO SEQ ID NO: 11:
    • (i) SEQUENCE MARKINGS:
      1. (A) LENGTH: 5109 base pairs
      2. (B) ART: nucleotide
      3. (C) STRING FORM: single strand
      4. (D) TOPOLOGY: linear
    • (ii) ART OF MOLECULAR: Genomic DNA
    • (ix) FEATURE:
      1. (A) NAME / KEY: exon
      2. (B) LOCATION: 1..5109
    • (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:
      Figure imgb0024
      Figure imgb0025
      Figure imgb0026
      Figure imgb0027

Claims (14)

  1. The use of a transgenic C. elegans which comprises a transgene which contains
    a) a nucleotide sequence which encodes the amyloid precursor protein (APP) or a part thereof,
    with the nucleotide sequence comprising the part of the APP nucleotide sequence which encodes the complete Aß peptide, and
    with the nucleotide sequence not being identical to the part of the APP which encodes a complete Aß peptide,
    b) where appropriate, one or more further coding and/or non-coding nucleotide sequences, and
    c) a promoter for expression in a cell of the nematode Caenorhabditis elegans (C. elegans) for identifying a γ-secretase activity and/or an α-secretase activity in C. elegans.
  2. The use as claimed in claim 1, wherein the nucleotide sequence encodes the 100 carboxyterminal amino acids of APP, beginning with the sequence of the Aß peptide and ending with the carboxy terminal amino acid of APP (C100 fragment).
  3. The use as claimed in either of claims 1 and 2, wherein the transgene contains the nucleotide sequence SEQ ID NO.: 1.
  4. The use as claimed in one of claims 1 to 3, wherein the transgene contains an additional coding nucleotide sequence which is located at the 5' end of the nucleotide sequence which encodes APP or a part thereof.
  5. The use as claimed in claim 4, wherein the additional nucleotide sequence encodes a signal peptide or a part thereof.
  6. The use as claimed in either of claims 4 and 5, wherein the additional nucleotide sequence encodes the APP signal peptide (SP) having the amino acid sequence SEQ ID NO.: 9.
  7. The use as claimed in one of claims 4 to 6, which contains the nucleotide sequence SEQ ID NO.: 2.
  8. The use as claimed in one or more of claims 1 to 7, which contains the nucleotide sequence SEQ ID NO.: 3.
  9. The use as claimed in one of claims 1 to 8, which contains an additional noncoding nucleotide sequence.
  10. The use as claimed in claim 9, wherein the additional non-coding nucleotide sequence is derived from the 42 bp intron of the APP gene and exhibits the sequence SEQ ID NO.: 4.
  11. The use as claimed in either of claims 9 and 10, which contains the nucleotide sequence SEQ ID NO.: 5.
  12. The use as claimed in one of claims 1 to 11, which contains a constitutive promoter or a promoter which can be regulated.
  13. The use as claimed in one of claims 1 to 12, wherein the promoter is active in neuronal, muscular or dermal tissue of C. elegans or is ubiquitously active in C. elegans.
  14. The use as claimed in one of claims 1 to 13, which contains a promoter which is selected from the group of the C. elegans promoters unc-54, hsp16-2, unc-119, G0A1 and sel-12.
EP99947485A 1998-10-24 1999-10-09 Transgenic c. elegans as a model organism for research into alzheimer's disease Expired - Lifetime EP1123388B1 (en)

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DE19849073A DE19849073A1 (en) 1998-10-24 1998-10-24 Transgene encoding amyloid precursor protein or its fragment, used to produce transgenic nematodes used, e.g. to screen for agents for treating Alzheimer's disease
PCT/EP1999/007578 WO2000024880A1 (en) 1998-10-24 1999-10-09 Transgenic c. elegans as a model organism for research into alzheimer's disease

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GB2357767B (en) * 1998-09-24 2002-08-21 Upjohn Co Alzheimer's disease secretase app substrates therefor and uses therefor
DE19856261C1 (en) 1998-12-07 2000-03-30 Hoechst Marion Roussel De Gmbh Detection of gamma-secretase by detection of A-beta peptide useful for determining gamma-secretase activity and for identifying inhibitors
DE19910108C2 (en) * 1999-03-08 2001-02-22 Falk Fahrenholz Cells that co-express an amyloid precursor protein and an alpha-secretase, a test procedure for the identification of alpha-secretase-active substances and one for the identification of further secretases, a test procedure for determining the susceptibility to Alzheimer's disease and the use of nucleic acids that code for an alpha-secretase , for gene therapy
DE10008315A1 (en) * 2000-02-23 2001-09-06 Metabowerke Kg Electric hand tool
US20030215896A1 (en) * 2001-04-25 2003-11-20 Yueming Li Gamma secretase substrates and in vitro assays
DE10130166A1 (en) * 2001-06-22 2003-01-16 Elegene Ag I Ins Identification of ses-1 and its uses
EP1429600B1 (en) * 2001-09-24 2008-12-31 Evotec AG An invertebrate animal model with alzheimer-like pathology for screening and testing molecules
US7491810B2 (en) * 2001-11-30 2009-02-17 U.S. Department Of Veterans Affairs Transgenic screen and method for screening modulators of brain-derived neurotrophic factor (BDNF) production
US7615676B2 (en) * 2001-11-30 2009-11-10 U.S. Department Of Veterans Affairs Transgenic screen and method for screening modulators of brain-derived neurotrophic factor (BDNF) production
EP1481987A1 (en) * 2003-05-26 2004-12-01 Aventis Pharma Deutschland GmbH Method for screening inhibitors of the gamma-secretase
ATE517913T1 (en) * 2004-01-28 2011-08-15 Sanofi Aventis Deutschland CAENORHABDITIS ELEGANS P21-ACTIVATED KINASE (PAK) AND ASSOCIATED LOSS OF FUNCTION PHENOTYPES
AU2008297070A1 (en) * 2007-09-12 2009-03-19 Probiodrug Ag Transgenic mice
WO2011017319A1 (en) 2009-08-03 2011-02-10 University Of Pittsburgh - Of The Commonwealth System Of Higher Education Methods of treating disorders associated with protein polymerization
US9072772B2 (en) 2009-11-05 2015-07-07 University of Pittsburgh—of the Commonwealth System of Higher Education Methods of treating disorders associated with protein aggregation
US8809617B2 (en) * 2009-11-05 2014-08-19 The University of Pittsburgh—Of the Commonwealth System of Higher Education Automated high-content live animal drug screening using C. elegans
WO2012178183A1 (en) * 2011-06-24 2012-12-27 Board Of Regents, The University Of Texas System Human age-related neurodegenerative nematode model and methods
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US5849999A (en) * 1996-10-16 1998-12-15 The Mclean Hospital Corporation Transgenic non-human mice expressing Flag-APP-C100 protein develop alzheimer's disease brain morphology and behavior
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